Methyl butenol synthase

ABSTRACT

The present invention provides novels genes encoding methyl butenol (MBO) synthase, methy butenol synthases and their use in methyl butenol production.

RELATED APPLICATION

This application is a U.S. National Stage Filing under 35 U.S.C. 371from International Application No. PCT/US2010/035820, filed May 21, 2010and published in English as WO 2010/135674 on Nov. 25, 2010, and claimspriority from U.S. Provisional Application Ser. No. 61/180,757 filed May22, 2009, which applications are herein incorporated by reference intheir entireties.

STATEMENT OF GOVERNMENT RIGHTS

This work was funded by NSF Grant No. IOS 0830225. The government hascertain rights to this invention.

BACKGROUND OF THE INVENTION

In 2007, there were 1.8 million alternative fuel vehicles sold in theUnited States, indicating an increasing popularity of alternative fuels.There is growing perceived economic and political need for thedevelopment of alternative fuel sources due to general environmental,economic, and geopolitical concerns of sustainability.

The major environmental concern is that most of the observed increase inglobally averaged temperatures since the mid-20th century is due to theobserved increase in greenhouse gas concentrations. Since burning fossilfuels is known to increase greenhouse gas concentrations in theatmosphere, it is believed that they are a likely contributor to globalwarming.

Although fossil fuels have become the dominant energy resource for themodern world, alcohol has been used as a fuel throughout history. Thefirst four aliphatic alcohols (methanol, ethanol, propanol, and butanol)are of interest as fuels because they can be synthesized and they havecharacteristics which allow them to be used in current engines.Biobutanol has an energy density that is closer to gasoline than theother alcohols; however, this advantage is outweighed by disadvantages(compared to ethanol and methanol) concerning, for example, production.

SUMMARY OF THE INVENTION

The present invention provides for the first time genes encoding methylbutenol (MBO) synthase and its use in methyl butenol production. Thus,one embodiment provides an isolated and purified methyl butenol (MBO)synthase nucleic acid molecule, wherein the MBO synthase nucleic acidmolecule comprises any one of SEQ ID NOs:1-17 or a nucleic acid moleculehaving at least about 80% sequence identity thereof.

Another embodiment provides an expression vector comprising a MBOsynthase nucleic acid molecule. One embodiment provides a prokaryotic oreukaryotic host cell transformed with a MBO synthase nucleic acidmolecule (e.g., in an expression vector). In one embodiment thetransformed host cells express the exogenous MBO synthase (mRNA orprotein). In one embodiment, the host cells express MBO synthase andyield MBO.

One embodiment provides an isolated and purified methyl butenol (MBO)synthase polypeptide, wherein the MBO synthase polypeptide comprises anyone of SEQ ID NOs:18-34 or a bioactive polypeptide having at least about80% sequence identity thereof.

Another embodiment provides a method to produce methyl butenolcomprising transforming a host cell with a nucleic acid molecule codingfor a methyl butenol synthase and expressing said molecule in a hostcell so as to yield methyl butenol. In one embodiment, the hose cellsare fermentative organisms, such as a bacteria, cyanobacteria or yeast(e.g., a bacteria or yeast that can break down sugar into alcohol) oreukaryotic micro algae. In one embodiment the fermentative organism isSaccharomyces cerevisiae, Klebsiella oxytoca, Synechococcus sp.,Synechocystis sp., Anabaena sp., Chlorella sp. Scenedesmus sp.,Bracteococcus sp. Chlamydomonus sp., C5- or C6-fermentative organisms(including Zymomonas (e.g., Zymomonas mobilis)) or a combinationthereof. In one embodiment, the transformed host cell is used infermentation with a carbohydrate to yield MBO (similar to bioethanolproduction).

In one embodiment, the MBO synthase (in a purified or unpurified form)is used in combination with a carbohydrate to yield MBO. In oneembodiment, the fermentative organism is bacteria, cyanobacteria oryeast (e.g., a bacteria or yeast that can break down sugar into alcohol)or eukaryotic micro algae. In one embodiment the fermentative organismis Saccharomyces cerevisiae, Klebsiella oxytoca, Synechococcus sp.,Synechocystis sp., Anabaena sp., Chlorella sp. Scenedesmus sp.,Bracteococcus sp. Chlamydomonus sp., C5- or C6-fermentative organisms(including Zymomonas (e.g., Zymomonas mobilis)) or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides nucleic acid molecules coding for a methyl butenolsynthase (SEQ ID NOs:1-17).

FIG. 2 provides the sequence of several methyl butenol synthases. (SEQID NO:18-34).

FIGS. 3A-B demonstrate the methylbutenol and isoprene production fromsoluble and insoluble extract fractions.

FIG. 4 demonstrates the effect of boiling and EDTA on enzyme activity.

FIG. 5 demonstrates the effect of co-factor replacement on MBO synthaseactivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the first time genes encoding methylbutenol (MBO) synthase and its use in methyl butenol production. Inparticular, the methyl butenol synthase gene was isolated from severalspecies of pine. The gene is useful for the enzymatic production ofmethyl butenol, which can be used as an alternative fuel (e.g., abiofuel or gasoline replacement). Thus, the methods provided hereinprovide a practical, non-polluting technology for producing methylbutenol using methyl butenol synthase proteins and genes. Exemplary cDNAsequences coding for methyl butenol synthases are provided in SEQ IDNOs:1-17, while exemplary amino acids sequences are provided in SEQ IDNOs:18-34 (see FIGS. 1 and 2).

Definitions

Certain terms used in the specification are defined and presented asfollows:

2-methyl-3-buten-2-ol (molecular formula of C₅H₁₀O; also known as methylbutenol or methylbutenol (MBO)) is a natural 5-carbon alcohol that isproduced and emitted from the foliage by several species of pine, e.g.,species in the genus Pinus, with the

Biosynthesis of MBO occurs in the chloroplast through the action of theenzyme MBO synthase, which uses DMADP derived from the MEP pathway as asubstrate.

“Associated with/operably linked” refers to two DNA sequences that arerelated physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operablylinked, or situated such that the regulator DNA sequence will affect theexpression level of the coding or structural DNA sequence.

“Coding DNA sequence:” refers to a DNA sequence that is translated in anorganism to produce a protein. “Expression” of a sequence includes theproduction of mRNA and/or protein.

“Isolated,” in the context of the present invention, an isolated nucleicacid molecule or an isolated polypeptide is a nucleic acid molecule orpolypeptide that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. Thus, the termisolated refers to a molecule (e.g., nucleic acid or protein) which isnot associated with one or more nucleic acid molecules, proteins or oneor more cellular components that are associated with the nucleic acidmolecule or protein in vivo. An isolated nucleic acid molecule or anisolated protein may exist in a purified form (e.g., ranges of purity insamples comprising isolated nucleic acid molecules or an isolatedprotein are 50-55%, 55-60%, 60-65% and 60-70%; ranges of purity alsoinclude 70-75%, 75-80%, 80-85%; 85-90%, 90-95%, and 95-100%; however,samples with lower purity can also be useful, such as about <25%,25-30%, 30-35%, 35-40%, 40-45% and 45-50%.) or may exist in a non-nativeenvironment such as, for example, a transgenic host cell.

A “cell” or “host cell” is a prokaryotic or eukaryotic cell.

Alternative fuels, also known as non-conventional fuels, are anymaterials or substances that can be used as fuels, other thanconventional fuels. Conventional fuels include: fossil fuels (petroleum(oil), coal, propane, and natural gas), and nuclear materials such asuranium. Some well known alternative fuels include biodiesel, bioalcohol(methanol, ethanol, butanol), chemically stored electricity (batteriesand fuel cells), hydrogen, non-fossil methane, non-fossil natural gas,vegetable oil and other biomass sources. For example, one alternative isalcohol fuel.

Alcohol fuels are usually of biological rather than petroleum sources.When obtained from biological sources, they are known as bioalcohols(e.g. bioethanol). As described herein 2-methyl-3-butene-2-ol (methylbutenol) can be manufactured in organisms carrying a synthase gene ofthe invention or by fermentative organisms in contact with a synthaseprotein of the invention and a carbohydrate source. It is a sustainableenergy resource that can provide a more environmentally and economicallyfriendly alternative to fossil fuels such as diesel and gasoline. It canbe combined with gasoline at different percentages, or can be used inits pure form. Generally, methyl butenol is more advantageous thanethanol as a fuel due to its increased density and relatively little orno water absorption as with ethanol. Unlike butanol, the presence of abranched carbon chain increases its octane rating and the presence of adouble carbon-carbon bond can reduce toxicity.

The terms “comprises,” “comprising,” and the like can have the meaningascribed to them in U.S. Patent Law and can mean “includes,” “including”and the like. As used herein, “including” or “includes” or the likemeans including, without limitation.

Methyl Butenol Synthase Genes/Proteins

Provided herein are methyl butenol synthase genes including those of SEQID NOs: 1-17 or a nucleic acid molecule which comprises a sequence thathas at least about 50%, at least about 60%, at least about 70%, about71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,about 78%, or about 79%, or at least about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, orabout 89%, or at least about 90%, about 91%, about 92%, about 93%, orabout 94%, or at least about 95%, about 96%, about 97%, about 98%, about99%, or about 100% sequence identity compared to any one of SEQ IDNOs:1-17 using one of the alignment programs available in the art usingstandard parameters.

Also provided herein are methyl butenol synthase proteins includingthose of SEQ ID NOs:18-34 or a polypeptide which comprises a sequencethat has at least about 50%, at least about 60%, at least about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 78%, or about 79%, or at least about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,or about 89%, or at least about 90%, about 91%, about 92%, about 93%, orabout 94%, or at least about 95%, about 96%, about 97%, about 98%, about99%, or about 100% sequence identity compared to any one of SEQ IDNOs:18-34 using one of the alignment programs available in the art usingstandard parameters. In one embodiment, the differences in sequence aredue to conservative amino acid changes. In one embodiment, thefunctional properties of the enzyme are improved through the use ofmolecular evolution and design studies.

Methods of alignment of sequences for comparison are available in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Computerimplementations of these mathematical algorithms can be utilized forcomparison of sequences to determine sequence identity. Suchimplementations include, but are not limited to: CLUSTAL in the PC/Geneprogram (available from Intelligenetics, Mountain View, Calif.); theALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTAin the Wisconsin Genetics Software Package, Version 8 (available fromGenetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).Alignments using these programs can be performed using the defaultparameters.

Expression of Methyl Butenol Synthase and Production of Methyl Butenol(MBO)

Methyl butenol synthase can be introduced into any number of organismsto cause them to produce methyl butenol synthase and/or methyl butenol.For example, the genes can be cloned into appropriate expression vectorsand expressed in bacteria or yeast. The creation of expression vectorscomprising the nucleic acid molecules of the invention and bacterial andyeast transformation techniques are described in detail in theliterature and available to an art worker. Typically an expressionvector contains (1) prokaryotic DNA elements coding for a bacterialreplication origin and an antibiotic resistance gene to provide for theamplification and selection of the expression vector in a bacterial hostor DNA elements to allow expression in a eukaryotic host cell (e.g.,yeast); (2) regulatory elements that control initiation of transcriptionsuch as a promoter; and (3) DNA elements that control the processing oftranscripts such as introns, transcription termination/polyadenylationsequence.

Methods to introduce a nucleic acid molecule into a vector are wellknown in the art (Sambrook et al., 1989). As an example, a vector intowhich the nucleic acid segment is to be inserted is treated with one ormore restriction enzymes (restriction endonuclease) to produce alinearized vector having a blunt end, a “sticky” end with a 5′ or a 3′overhang, or any combination thereof. The vector may also be treatedwith a restriction enzyme and subsequently treated with anothermodifying enzyme, such as a polymerase, an exonuclease, a phosphatase ora kinase, to create a linearized vector that has characteristics usefulfor ligation of a nucleic acid molecule into the vector. The nucleicacid molecule that is to be inserted into the vector is treated with oneor more restriction enzymes to create a linearized segment having ablunt end, a “sticky” end with a 5′ or a 3′ overhang, or any combinationthereof. The nucleic acid molecule may also be treated with arestriction enzyme and subsequently treated with another DNA modifyingenzyme. Such DNA modifying enzymes include, but are not limited to,polymerase, exonuclease, phosphatase or a kinase, to create apolynucleic acid segment that has characteristics useful for ligation ofa nucleic acid molecule into the vector.

The treated vector and nucleic acid molecule are then ligated togetherto form a construct containing a nucleic acid segment according tomethods known in the art (Sambrook, 2002). Briefly, the treated nucleicacid molecule and the treated vector are combined in the presence of asuitable buffer and ligase. The mixture is then incubated underappropriate conditions to allow the ligase to ligate the nucleic acidmolecule into the vector.

An example of the preparation of an expression vector and expression ofMBO synthase in an organism is described in the examples below. However,any expression vector and organism combination for expression availableto an art worker may be acceptable for use in the methods of theinvention. It will be clear to one of ordinary skill in the art whichvector should be used depending on which cell type is used for a hostcell.

Alternatively, the methyl butenol synthases of the invention can also bechemically synthesized through peptide synthesis procedures available tothe art. The free enzyme can be contacted with an appropriate substrate(a source of carbohydrate) to produced methyl butenol (with our withoutthe use of a host cell).

After preparation of the expression vector and introduction into anappropriate host cell, the cells can be grown in culture to producemethyl butenol synthase and/or methyl butenol, which can be collected(and optionally distilled or condensed).

Alternatively, the culture can be used to produce the free enzyme whichis then used to produce MBO. For example, a source of carbohydrate(e.g., sugars (e.g., C_(n)H_(2n)O_(n) (n is between 3 and 7), includinga monosaccharide (e.g., glucose, dextrose or fructose) or a disaccharide(e.g., sucrose), starches (e.g., a carbohydrate consisting of a largenumber of glucose units joined together by glycosidic bonds, such asamylase or amylopectin) and/or cellulose (e.g., an organic compound withthe formula (C₆H₁₀O₅)_(n), a polysaccharide consisting of a linear chainof several hundred to over ten thousand β(1→4) linked D-glucose units))and a methyl butenol (MBO) synthase of the invention can be combined toyield MBO, which can be purified further (e.g., distilled or condensedfrom the head space).

The MBO produced by the methods of the invention can be used as analternative fuel source.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andis not to be construed as limiting the scope thereof.

Example 1 Preparation of Methyl Butanol Synthase vectors and Assay forActivity

Materials and Methods

RNA Extraction

Total RNA was extracted from frozen pine needle tissue from Pinussabiniana using a modified CTAB procedure adapted from (Chang et al.(1993) Plant Molecular Biology Reporter, 11(2):113-116.). Needle samples(250 mg) were ground under liquid nitrogen, and extracted for 45 minutesat 70° C. in 800 u1 volumes of extraction buffer containing 2% CTAB, 2%PVP-40, 100 mM Tris pH8.0, 25 mM EDTA, 2.0M NaCl, and 0.5 g/L Spermidinewith 4% β-mercaptoethanol, 4% PVPP, and an additional 4% PVP40 was addedjust before use. Following incubation at 70° C. for 45 minutes, sampleswere extracted twice with 5000 volumes of 24:1 (v:v) chloroform:isoamylalcohol solution. RNA was then precipitated by adding ¼ volume of 10MLiCl to the aqueous phase, gently mixing, and storing the mixtureovernight at 4° C. Following centrifugation to collect the RNA pellet,the supernatant was discarded and the pellet was re-suspended in 500 μlof SSTE Buffer containing 1.0M NaCl, 0.5% SDS, 10 mM Tris pH8.0, and 1mM EDTA. This solution was then extracted once with equal volumes ofphenol, once with phenol:chloroform:IAA (24:24:1 v/v), and once withchloroform:IAA (24:1 v/v). To precipitate the RNA pellet, 2 volumes ofcold (−20° C.) ethanol were added to the supernatant followed byincubation for 2 hours at −20° C. Following centrifugation to collectthe pellet, the RNA was air dried, re-suspended in distilled water, andstored at −20 C until further use.

cDNA Synthesis

First strand cDNA synthesis was performed using M-MLV reversetranscriptase obtained from Invitrogen (Carlsbad, Calif.) following themanufacturers instructions. Total RNA (2 μg), and 1 μg oligo-dT(17)primer were denatured by incubation at 70° C. for 5 minutes, and thenplaced on ice for 5 min to anneal the oligo-dT primer to the mRNA polyAtail. Reagents were added to this mixture to achieve a 50 μL volumecontaining 1× concentration M-MLV buffer, 0.5 mM dNTPs, 0.1M DTT and 2.5units of RNasin (Promega, Madison Wis.). The first strand synthesisreactions were performed by incubating for 60 min at 37° C., followed byincubating for 10 min at 42° C., and heat inactivation of the enzyme byincubating at 70° C. for 15 min. Reactions were cooled on ice and storedat −20° C. until further use.

Primer Design

Initially, degenerate PCR primers were designed to correspond to highlyconserved regions of known pine monoterpene synthases. Sequencing of thePCR amplified products revealed 3 distinct sequences which showed highsimilarity to known pine monoterpene synthases. One sequence contained aphenylalanine residue 7 amino acids upstream of the highly conservedDDXXD motif of terpene synthases. Since this position corresponds to thelocation of a phenylalanine residue in isoprene synthases and isbelieved to limit the size of the substrate that can fit into theenzymes active site, this phenylalanine containing sequence was selectedfor further exploration. To obtain the 5′ and 3′ ends of this sequence5′ and 3′ rapid amplification of cDNA ends (RACE) was employed. Forthese procedures gene specific forward and reverse primers were designedfrom the previously selected partial MBO synthase sequence.

PCR Reactions

Initial PCR reactions were run using 1.25 units of Gotaq DNA polymerasein 30 μl reaction volumes containing 1× Gotaq buffer, 20 μM dNTPs, 1 μMconcentrations of each primer, and 0.2 μg template cDNA. In preparationfor cloning, full length cDNAs were amplified using either Pfu turbo(Stratagene, La Jolla Calif.) or Platinum Pfx (Invitrogen, CarlsbadCalif.) DNA polymerases according to the manufacturer instructions. Toamplify full length cDNAs PCR reactions were run using an initialdenaturation of 2 min at 95° C., followed by 35 repetitions ofdenaturation at 95° C. for 30 sec, annealing at 52° C. for 45 sec, andextension at 72° C. for 3 min, followed by a final extension at 72° C.for 9 min.

Rapid Amplification of cDNA Ends (RACE)

Rapid amplification of cDNA ends (RACE) procedures were employed toamplify the 5′ and 3′ ends of the partial MBO sequence obtained usingdegenerate primers. RACE reactions were run using 1.25 units of GotaqDNA polymerase in 30 μl reaction volumes containing 1× Gotaq buffer, 20μM dNTPs, 1 μM concentrations of each primer (gene specific, adapter,oligo dt-adapter), and 0.2 μg template cDNA.

Cloning into the Expression Vector

The full length amino acid sequence of the MBO synthase gene contains anN-terminal region similar to the plastid transport sequences found inknown conifer monoterpene synthases. These transport sequences have beenfound to interfere with protein expression in E. coli (Williams et al.(1998) Biochemistry, (37):12213-12220), hence primers were designed toamplify a fragment of the putative MBO synthase gene lacking thischloroplast transport sequence. Hereafter this will be referred to as anexpression length sequence.

Expression length sequences were first ligated into the vector p-GEMTeasy (Promega, Madison Wis.) and transformed into the E. coli hoststrain DH5α. pGEMT-easy plasmids containing the MBO synthase gene(pGEMT-MBO) were extracted from the DH5α host and digested with therestriction enzymes SphI and PstI to release an ˜1800 bp fragmentcontaining the MBO synthase gene with a 5′ SphI and 3′ PstI splice site.This fragment was then gel purified and ligated into the expressionvector pQE-31 (Qiagen) following digestion of pQE-31 vector with therestriction enzymes SphI and PstI and gel purification of the 3.4 kbdigested product. Gel purification of restriction digests was done usinga Qiagen gel purification kit (Qiagen). Following ligation of theexpression length MBO sequence into the expression vector pQE-31,pQE-31-MBO was transformed into E. coli strain BL21-CodonPlus(DE3)-RIL(Stratagene, La Jolla Calif.). This strain of E. coli has been modifiedto correct for codon bias by increasing the expression of several t-RNAsthat match codons that are common in plant terpene synthases, but rarein E. coli. The MBO synthase gene contains several codons for whicht-RNAs in E. coli are rare.

Functional Expression and Assay for Methyl Butenol Production

E. coli BL21-Codon Plus(DE3)-RIL containing expression plasmidpQE-31-MBO were grown overnight at 37° C. with shaking at 200 rpm in 5ml Luri Broth supplemented with 200 μg/ml ampicillin and 34 μg/mlchloramphenicol. Aliquots of bacterial culture (0.5 ml) were placed into5 ml vials and protein expression induced by adding 0.5 mM IPTG. Vialswere capped with a septum and incubated at 30° C. with shaking at 200rpm for 2 hours before analysis of headspace volatiles by gaschromatography.

To measure methyl butenol in vial headspace, 3 ml aliquots of headspacewere withdrawn from the vial and cryofocused by injection into astainless steel sample loop immersed in liquid nitrogen. The cryofocusedsample was then flash vaporized by immersing the sample loop in warmwater and flushed onto a widebore capillary DB-1 GC column, 30 m length,0.32 mm ID, 5 micron film (JW Scientific, Folsom, Calif.) using flow ofHelium carrier gas, and analyzed using a Photoionization detector.Component separation was achieved using a temperature program of 30° C.for 10 minutes, followed by a temperature ramp of 2° C. per minute and ahold at 60° C. for 5 minutes. The identity of methylbutenol measured invial headspace was assessed by comparison of GC peak retention timeswith those obtained from authentic MBO standards, and by GC-massspectrometry.

Example II Assays of Activity of MBO Synthase from P. sabiniana

The below assays were run at pH 8.0 in a 50 mM Hepes buffer with 10%glycerol, 100 mM KCl, 20 mM MgCl₂, 5 mM MnCl₂, and 10 mM DMAPP. Assayswere incubated for 2 hours at 37° C., and vial headspace was run on GC.Products were identified by comparing retention times with authenticstandards.

A) Methylbutenol and Isoprene Production from Soluble and InsolubleExtract Fractions

FIG. 3 demonstrates that expressed enzyme (with both N and C-terminalHis tags (for purification) possess dual catalytic activity and can formboth isoprene and methylbutenal from the substrate DMAPP (dimethylallylpyrophosphate (or -diphosphate)). The enzyme partitions to the insolublefraction during the extraction process.

B) Effect of Boiling and EDTA on Enzyme Activity

FIG. 4 demonstrates that most of the activity is in the insolublefraction. Furthermore, experiments determined that the conversion ofDMAPP to isoprene and MBO is the result of the MBO synthase. Forexample, assays run with a boiled pellet (the most active extract) showlittle or no production of isoprene or MBO. Thus, boiling the extractinactivated the enzyme responsible for DMAPP conversion. Also, assaysrun with active enzyme in the presence of EDTA show little conversion ofDMAPP to isoprene or MBO. This is significant because MBO synthaserequires a divalent cation to function. EDTA chelates these cations andthus eliminated enzyme function.

C) Effect of Co-Factor Replacement on MBO Synthase Activity

FIG. 5 demonstrates the effect of selectively removing the cationco-factors for MBO synthase function. The plant extract MBO synthaseuses both Mn²⁺ and K⁺ as co-factors. Angiosperm isoprene synthases usesMg²⁺. By washing the pellet containing the recombinant MBO synthaseprotein extracted from E. coli, the Mn²⁺ and K⁺ ions were removed. Thepellets were then resuspended in isoprene synthase buffer contaning Mg²⁺or MBO synthase buffer containing Mn²⁺ and K. Assays run in isoprenesynthase buffer (without Mn²⁺ and K⁺ cofactors) show little activity.Assays run in MBO synthase buffer (with both Mn²⁺ and K⁺) show activityproducing isoprene and MBO with about 3 fold preference for forming MBO.Thus, the catalytic activity observed is from an MBO synthase (e.g., thecloned gene).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

What is claimed is:
 1. An isolated and purified methyl butenol (MBO) synthase cDNA molecule, wherein the MBO synthase cDNA molecule comprises SEQ ID NO:8 or a nucleic acid molecule having at least 90% sequence identity thereof.
 2. An expression vector comprising a MBO synthase cDNA molecule of claim
 1. 3. An isolated prokaryotic or eukaryotic host cell transformed with a MBO synthase cDNA molecule of claim
 1. 4. A prokaryotic or eukaryotic host cell transformed with the vector of claim
 2. 5. The host cell of claim 3, wherein the transformed cell expresses MBO synthase.
 6. A method to produce methyl butenol comprising transforming a host cell with a cDNA molecule of claim 1 and expressing said molecule in a host cell so as to yield methyl butenol.
 7. The method of claim 6, wherein said host cell is a strain of yeast, bacteria, cyanobacteria, eukaryotic micro algae or a combination thereof.
 8. The method of claim 7, wherein said yeast is Saccharomyces cerevisiae.
 9. The method of claim 7, wherein said bacteria is a C5- or C6-fermentative organism.
 10. The method of claim 9, wherein said C5-fermenative organism is a species of Zymomonas.
 11. The method of claim 7, wherein said bacteria is Klebsiella oxytoca.
 12. The method of claim 7, wherein said cyanobacteria is Synechococcus sp., Synechocystis sp., or Anabaena sp.
 13. The method of claim 7, wherein said eukaryotic micro algae is Chlorella sp. Scenedesmus sp., Bracteococcus sp. or Chlamydomonus sp.
 14. An isolated and purified methyl butenol (MBO) synthase cDNA, wherein the MBO synthase cDNA comprises SEQ ID NO:8. 